KR20230147758A - Immune adjuvant for cancer - Google Patents

Abstract

The present invention relates to a medicine for treating cancer or infectious diseases comprising a combination of an immune checkpoint inhibitor and an adjuvant composition. More specifically, it relates to a medicine for cancer or infectious disease, characterized by the combined use of an anti-PD-1 antibody or anti-PD-L1 antibody and a nucleic acid adjuvant consisting of double-stranded RNA and single-stranded ODN.

Description

IMMUNE ADJUVANT FOR CANCER}

[Related applications]

This application claims priority based on Japanese Patent Application Nos. 2016-147657 (filed on July 27, 2016) and 2017-079445 (filed on April 13, 2017), the contents of which are included in this specification. It is incorporated by reference.

[Technical field]

The present invention relates to the combined use of an immune checkpoint inhibitor and an adjuvant composition. More specifically, it relates to a new treatment for cancer or infectious diseases characterized by the combined use of an anti-PD-1 antibody or an anti-PD-L1 antibody and a nucleic acid adjuvant consisting of double-stranded RNA and single-stranded ODN.

The living body is equipped with a natural immune defense system that excludes when cells become cancerous or when pathogens such as bacteria or viruses invade the body, and the molecules that control the immune defense system are called immune checkpoints. It is thought that cancer cells often cleverly avoid the surveillance of the immune defense system by enhancing the expression of “immune checkpoints.”

PD-1 and PD-L1 are representative immune checkpoint molecules, and antibodies targeting these molecules have been confirmed to exhibit significant anticancer activity in various types of cancer, and are currently being developed as pharmaceuticals. However, with respect to PD-1/PD-L1 antibody therapy, there are reports showing remission in more than 50% of metastatic melanomas, while remission in solid tumors remains at 20 to 30%, and its main efficacy is sufficient. It cannot be said that it is. In addition, there are patients who respond to PD-1 antibody therapy and patients who do not respond, and there is a need to differentiate them and develop treatments for patients who do not respond.

Anticancer immune vaccine therapy is a fourth treatment for cancer that cannot be treated with surgery, anticancer drugs, or radiation therapy, and has been receiving particular attention in recent years. Side effects are often a problem in anticancer drug discovery, but immunovaccine therapy has the advantage of less invasion and side effects because cancer cell-specific antigens are used to selectively attack cancer cells. In immunovaccine therapy, adjuvants are often used to assist and enhance the effectiveness of the vaccine by enhancing antigenicity.

Activation of Toll-like receptor (TLR) causes a natural immune response in dendritic cells, leading to cytokine production or activation of cellular immunity. Double-stranded RNA such as poly(I:C) has been shown to be a promising vaccine adjuvant because it becomes a TLR3 ligand and exhibits a strong anti-cancer effect, but clinical application has been abandoned due to side effects such as inflammation and cytokinesis.

The inventors discovered a GpC dinucleotide-containing oligodeoxynucleotide (GpC ODN) that inhibits poly(I:C) induction of IFN-β expression through TLR3, and reported that this GpC ODN was introduced into cells through TLR3. (Non-patent Document 1). In addition, it has been reported that diRNA (defective interference RNA) derived from measles virus has an adjuvant function (Patent Document 1).

Additionally, the inventors discovered that the nucleic acid designed based on the above-mentioned GpC ODN and diRNA reaches TLR3 on endsomes and has strong adjuvant activity (Patent Document 2). This adjuvant does not induce excessive production of inflammatory cytokines and induces NK/CTL-dependent anticancer activity in a mouse transplant cancer model (Patent Document 3 and Non-Patent Document 2), making it a non-inflammatory nucleic acid with few side effects. As an immune adjuvant, application to anti-cancer immune vaccines is expected.

WO2008/065752 WO2012/014945 WO2016/088784

Itoh et al., The Journal of Immunology, 2008, vol.181, No.8, pp5522-5529 Matsumoto et al., Nature Communications, 2015, 6: 6280

The object of the present invention is to provide a new cancer immunotherapy by improving the main efficiency of PD-1/PD-L1 antibody therapy while maintaining safety.

In order to solve the above problems, by using a nucleic acid adjuvant consisting of double-stranded RNA and single-stranded oligodeoxynucleotide (single-chain ODN) in combination with tumors for which anti-PD-L1 antibodies alone are ineffective, CTL dependence can be reduced. It was confirmed that significant tumor regression can be induced.

The present invention was completed based on the above-mentioned knowledge and relates to the following (1) to (17).

(1) A medicine for treating cancer or infectious disease comprising a combination of an immune checkpoint inhibitor and an adjuvant composition, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody or an anti-PD-L1 antibody, and the adjuvant composition The anti-cant composition is a nucleic acid consisting of double-stranded RNA and a single-stranded oligodeoxynucleotide (single-stranded ODN) containing a sequence in which CpG is replaced by GpC, TpC, or CpC in a CpG ODN, or a partial sequence thereof of 5 bases or more in length. The drug, including an adjuvant (double-stranded RNA functions as a TLR3 ligand and single-chain ODN functions as a delivery molecule to the endosome);

(2) The medicine according to (1), wherein an immune checkpoint inhibitor and an adjuvant composition are used in combination;

(3) The medicine according to (2), wherein the immune checkpoint inhibitor and the adjuvant composition are administered simultaneously or sequentially;

(4) A nucleic acid matrix consisting of double-stranded RNA and a single-stranded oligodeoxynucleotide (single-stranded ODN) containing a sequence in which CpG is replaced with GpC, TpC, or CpC in a CpG ODN, or a partial sequence of 5 bases or more in length thereof. A medicine for treating cancer or infectious diseases containing an immune checkpoint inhibitor containing an anti-PD-1 antibody or an anti-PD-L1 antibody as an active ingredient, characterized in that it is used in combination with an adjuvant composition containing an anti-PD-1 antibody;

(5) The medicine according to any one of (1) to (4), wherein the adjuvant composition contains an antigen molecule selected from bacterial antigens, viral antigens and cancer antigens;

(6) The drug according to any one of (1) to (5), wherein the terminal of the nucleic acid constituting the nucleic acid adjuvant is not phosphorylated;

(7) The double-stranded RNA has a nucleotide sequence of at least 30 consecutive bases, preferably at least 40 bases, of the nucleotide sequence shown in SEQ ID NO: 1, and has a total length of 100 to 160 bases, preferably 110 to 150 bases. length, more preferably 120 to 140 bases in length, or a nucleic acid having 80% sequence identity with the above sequence and having TLR3 activation ability. Medications listed;

In the nucleic acid, the double-stranded RNA is a nucleic acid containing a contiguous 30 to 80 base long nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 repeated 2 to 4 times, preferably a contiguous nucleotide sequence shown in SEQ ID NO: 1. Nucleic acids containing a base sequence of 30 to 50 bases in length repeated three times are included.

(8) The double-stranded RNA is a nucleic acid having a base sequence shown in any of SEQ ID NOs: 2 to 11, or a nucleic acid having a contiguous base sequence of at least 100 bases, preferably at least 110 bases, more preferably at least 120 bases, or the medicine according to any one of (1) to (7), which is a nucleic acid that has 80% sequence identity with the above sequence and has TLR3 activation ability;

(9) The double-stranded RNA is a nucleic acid having the base sequence shown in SEQ ID NO: 11, or a nucleic acid having a contiguous base sequence of at least 100 bases, preferably at least 110 bases, more preferably at least 120 bases, or the above sequence The drug according to any one of (1) to (8), which is a nucleic acid having 80% sequence identity and TLR3 activation ability;

(10) The medicine according to any one of (1) to (9), wherein the single-chain ODN is a nucleic acid comprising the base sequence shown in any of SEQ ID NOs: 19 to 39 or a partial sequence having a length of 5 bases or more;

(11) The medicine according to any one of (1) to (9), wherein the single-chain ODN is a nucleic acid comprising the base sequence shown in any of SEQ ID NOs: 19 to 24 or a partial sequence thereof having a length of 5 bases or more;

(12) The drug according to any one of (1) to (11), wherein the single-chain ODN is 15 to 28 bases long;

(13) The drug according to any one of (1) to (12), wherein the nucleotides constituting the single-stranded ODN are modified with phosphorothioate;

(14) The nucleic acid adjuvant is a nucleic acid containing the sense strand shown in SEQ ID NO: 40 and the antisense strand shown in SEQ ID NO: 41, a nucleic acid containing the sense strand shown in SEQ ID NO: 42 and the antisense strand shown in SEQ ID NO: 43, or the above. The drug according to any one of (1) to (13), which is a nucleic acid comprising a sense strand and an antisense strand each having 80% sequence identity with the sequence and having adjuvant activity;

(15) nucleic acid adjuvant,

1) It contains a sense strand represented by SEQ ID NO: 40 and an antisense strand represented by SEQ ID NO: 41, or

2) It contains a sense strand represented by SEQ ID NO: 42 and an antisense strand represented by SEQ ID NO: 43, or

3) It contains a sense strand represented by SEQ ID NO: 44 and an antisense strand represented by SEQ ID NO: 45, or

4) Comprising a sense strand represented by SEQ ID NO: 46 and an antisense strand represented by SEQ ID NO: 47,

The medicine according to any one of (1) to (14).

(16) A nucleic acid matrix consisting of double-stranded RNA and a single-stranded oligodeoxynucleotide (single-stranded ODN) containing a sequence in which CpG is replaced with GpC, TpC or CpC in CpG ODN, or a partial sequence of 5 bases or more in length thereof. An adjuvant composition comprising an adjuvant,

The double-stranded RNA is a nucleic acid comprising a contiguous 30 to 80 base sequence of the nucleotide sequence shown in SEQ ID NO: 1 repeated 2 to 4 times, preferably a contiguous 30 to 50 nucleotide sequence shown in SEQ ID NO: 1. An adjuvant composition, which is a nucleic acid containing a base-length base sequence repeated three times.

(17) A medicine for treating cancer or an infectious disease, comprising the adjuvant composition according to (16).

According to the present invention, the effect or main efficiency of PD-1/PD-L1 antibody therapy can be improved. The adjuvant of the present invention is efficiently introduced into dendritic cells, specifically activates TLR3 and induces NK/CTL without inducing production of systemic inflammatory cytokines. Therefore, unlike other immune combination therapies, it does not cause side effects such as cytokineemia and mildly improves the effect of PD-1/PD-L1 antibodies, resulting in tumor regression.

Figure 1a shows the induction of expression of tumor-specific CTL in the spleen and subgenual lymph nodes of mice (wild type (WT) and various knockout (KO) mice) administered subcutaneously with PBS (control), OVA, and OVA+ARNAX ( %).
Figure 1b shows the production of IFN-γ (pg/ml) in spleen cells of mice (wild type (WT) and various knockout (KO) mice) administered subcutaneously with PBS (control), OVA, and OVA+ARNAX. indicates.
Figure 1c shows tumor-specific CTL derived from the spleen of mice (wild type (WT) and various knockout (KO) mice) administered subcutaneously with PBS (control), OVA, and OVA+ARNAX, analyzed by flow cytometry. Shows the results.
Figure 2 shows the induction of cytokines when raw (control), poly(I:C), and ARNAX were administered subcutaneously (a) and intravenously (b).
Figure 3a shows PD-L1 expression in EG7 cell line. Figure 3b shows an outline of the experimental protocol of Example 3 (antitumor effect by administration of ARNAX and antigen). Figure 3c shows the change in tumor volume over time in mice subcutaneously implanted with EG7 cells on Day 0. On Day 7, significant tumor regression was observed in mice administered OVA+ARNAX subcutaneously (-●-), but tumor regression was not observed in mice administered PBS (control) (-■-).
Figure 3d shows CD8-positive T cells (left), OVA tetramer-positive/CD8-positive T cells (middle), CD11c-positive/in the tumor of mice administered subcutaneously with PBS (control) or OVA+ARNAX. The percentage of CD8 positive T cells (right) is shown. Figure 3E shows the results of quantitative PCR analysis of the induction of expression of Gzmb, Ifng, Prf1, Cxcl9, Cxcl10, and Cxcl11 in mice subcutaneously administered PBS (control) or OVA+ARNAX.
Figure 3f shows immunostaining for DAPI and CD8α in tumor tissues of mice subcutaneously administered PBS (control) or OVA+ARNAX.
Figure 4a shows a comparison of PD-L1 expression between the PD-L1 high-expressing EG7 cell line (solid line) and the normal cell line (dashed line). Figure 4b shows an outline of the experimental protocol of Example 4 (combined use of ARNAX and anti-αPD-L1 antibody). Figure 4c shows changes over time in the tumor volume of mice that were subcutaneously transplanted with EG7 cells on Day 0, subcutaneously administered with PBS (control) or OVA+ARNAX on Day 7, and intravenously administered anti-mouse PD-L1 antibody or isotype antibody. represents. Tumor regression was not observed when PD-L1 antibody was administered alone (-■-), but significant tumor regression was seen when PD-L1 antibody was administered in combination with OVA+ARNAX (-x-), and the effect was similar to that of OVA+ARNAX alone ( It is higher than -▲-).
Figure 4d shows CD8 positive T cells (left) in the spleen and tumor of mice administered intravenously with anti-mouse PD-L1 antibody or isotype antibody after subcutaneous administration of PBS (control) or OVA+ARNAX, and OVA tetrad. The proportions of body positive/CD8 positive T cells (middle left), CD11c positive/CD8 positive T cells (middle right), and PD-1 positive/CD8 positive T cells (right) are shown (top in spleen, bottom in tumor). .
Figure 4E shows the levels of Gzmb, Ifng, Prf1, Cxcl9, Cxcl10, and Cxcl11 in the tumor of mice administered intravenously with anti-mouse PD-L1 antibody or isotype antibody after subcutaneous administration of PBS (control) or OVA+ARNAX. The results of analysis of expression induction by RT-PCR are shown.
Figure 5 shows the mechanism of action of the adjuvant nucleic acid (ARNAX) and anti-PD-L1 antibody according to the present invention.
Figure 6a shows PD-L1 expression in MO5 cell line (solid line). Figure 6b shows an outline of the experimental protocol of Example 5 (combined use of ARNAX and anti-αPD-L1 antibody). Figure 6c shows changes in tumor volume over time in mice that were subcutaneously transplanted with MO5 cells on Day 0, subcutaneously administered with PBS (control) or OVA+ARNAX on Day 10, and intravenously administered with anti-mouse PD-L1 antibody or isotype antibody. represents. Compared to PD-L1 antibody administration alone (-■-) and OVA+ARNAX administration (-▲-), significantly higher tumor regression is seen in PD-L1 antibody and OVA+ARNAX combined administration (-X-). Figure 6d shows CD8 positive T cells (left) in the spleen and tumor of mice administered intravenously with anti-mouse PD-L1 antibody or isotype antibody after subcutaneous administration of PBS (control) or OVA+ARNAX, and OVA tetrad. The proportions of body positive/CD8 positive T cells (middle left), CD11c positive/CD8 positive T cells (middle right), and PD-1 positive/CD8 positive T cells (right) are shown (top in spleen, bottom in tumor). . Figure 6E shows induction of expression of Gzmb, Ifng, Prf1, Cxcl9, and Cxcl10 in the tumor of mice administered intravenously with anti-mouse PD-L1 antibody or isotype antibody after subcutaneous administration of PBS (control) or OVA+ARNAX. Shows the results of analysis by RT-PCR.
Figure 6f shows the percentage of tumor-infiltrating immune cells. Proportion of tumor-infiltrating lymphocytes gated on CD45-positive cells (top), percentage of dendritic cell (DC) subsets gated on CD45-positive cells, percentage of tumor-infiltrating monocytes, CD11b-positive myeloid cell subsets, and macrophages gated on CD45-positive cells.
Figure 7 shows the structures of ARNAX120#1, ARNAX#2, ARNAX130, and ARNAX140.
Figure 8 shows the ability of ARNAX120#2 to activate the IFN-β promoter through TLR3.
Figure 9 shows the ability of ARNAX120#1 and ARNAX130 to activate the IFN-β promoter through TLR3.
Figure 10 shows the in vivo cross-priming activity (variable dose) of ARNAX140.
Figure 11 shows the in vivo cross-priming activity (variable dose) of ARNAX120#2.
Figure 12 shows the antitumor effect (EG7) of ARNAX120#1.

The present invention provides a medicine for treating cancer or infectious diseases, comprising a combination of an immune checkpoint inhibitor (anti-PD-1 antibody or anti-PD-L1 antibody) and a nucleic acid adjuvant consisting of double-stranded RNA and single-stranded ODN. It's about.

1. Immune checkpoint inhibitors

Molecules that control the natural immune defense system against pathogens such as cancer cells, bacteria, and viruses are called immune checkpoints, for example, PD-1 expressed on the surface of effector T cells, or PD-1 expressed on the surface of tumor cells. Examples include L1 or PD-L2, CTLA-4 expressed on the surface of activating T cells or suppressor T cells Tregs, and CD80 or CD86 expressed on the surface of dendritic cells. Among these, CTLA-4/CD80/CD86 functions in the priming phase in which sensitized T cells differentiate from naive T cells into T _H1 , T _H2 , T _FH cells, etc., and PD-1/PD-L1/PD- L2 functions in the effector phase, where effector cells attack tumor cells and the like.

“Immune check point inhibitor” refers to a substance or molecule that inhibits “immune check points” and controls the natural immune defense system. For example, antibodies specific to the above immune checkpoints (anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, etc.) can be cited as representative examples of “immune checkpoint inhibitors.”

The “immune checkpoint inhibitor” used in the present invention is an immune checkpoint inhibitor that functions in the effector phase, that is, a substance/molecule that inhibits PD-1/PD-L1/PD-L2. Preferably, the “immune checkpoint inhibitor” is an anti-PD-1 antibody or an anti-PD-L1 antibody.

“Anti-PD-1 antibody”

The “anti-PD-1 antibody” used in the present invention is not particularly limited as long as it can bind to PD-1 and inhibit its function as an immune checkpoint. As anti-PD-1 antibodies, there are those that are already approved and sold as pharmaceuticals or those that are under development, and these can be used suitably. Such “anti-PD-1 antibodies” include nivolumab (Opdivo (GSK/Yakuhing Ono)), pembrolizumab (MK-3475 (Merck)), a humanized IgG4 type antibody, and pidirizumab (CT-011 (CureTech) )), but is not limited to these.

“Anti-PD-L1 antibody”

Additionally, the “anti-PD-L1 antibody” used in the present invention is not particularly limited as long as it can bind to PD-L1 and inhibit its function as an immune checkpoint. As anti-PD-L1 antibodies, there are those that have already been approved (sold) as pharmaceuticals or those that are under development, and these can be used suitably. Such “anti-PD-L1 antibodies” include atezolizumab (MPDL3280A/RG-7446 (Roche/Jugai), durvalumab (MEDI4736 (AstraZeneca)), avelumab (MSB0010718C (Merck)), and MED10680. /AMP-514, but is not limited to these.

Antibodies may be produced based on conventional methods. In that case, since the antibody reduces heterologous antigenicity to humans, it is preferable that it is a chimeric antibody, humanized antibody, or fully human antibody. The antibody may be an antibody fragment as long as it has a function as an immune checkpoint inhibitor. Examples of antibody fragments include F(ab') ₂ , Fab', Fab, Fv, and scFv.

The “immune check point inhibitor” of the present invention may contain a pharmacologically acceptable carrier or additive. Such carriers and additives include, for example, surfactants, excipients, colorants, flavoring agents, preservatives, antioxidants, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity promoters, coagulants, etc. However, it is not limited to these, and other commonly used carriers may be appropriately used.

Specifically, aqueous carriers include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), vegetable oils such as olive oil, and organic esters such as ethyl oleic acid.

Non-aqueous carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylamino acetate, and polyvinyl methylcellulose. Examples include rolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc.

Antioxidants include the water-soluble antioxidants ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite, and the fat-soluble antioxidants ascorbyl palmitate, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). ), lecithin, propyl gallate, α-tocopherol, and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

The route of administration of the “immune checkpoint inhibitor” of the present invention is not particularly limited, but parenteral administration is preferred, and specific examples include injection, nasal administration, transpulmonary administration, and transdermal administration. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The administration method can be appropriately selected depending on the patient's age and symptoms.

The dosage of the “immune checkpoint inhibitor” of the present invention is appropriately determined depending on the purpose of use, route of administration, etc. Adjusted to result in the desired optimal response (e.g., therapeutic response). Since the active ingredient is an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, generally 0.01 to 5 mg/kg of patient body weight. For example, the dosage is about 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, or 10 mg/kg body weight, or ranges from 1 to 10 mg/kg. Typical treatment regimens include, for example, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 3 months. Or, it is administered once at intervals of 3 to 6 months. For example, in the case of intravenous administration, it is 1 mg/kg body weight or 3 mg/kg body weight. The number of administrations is appropriately set depending on the symptoms, and may be administered as a single bolus or divided into several administrations over time. For example, in some cases, 6 doses are administered at 4-week intervals, then at 3-week intervals, and in some cases, 3 mg/kg body weight is administered once, then 1 mg at 3-week intervals. In some cases, it is administered at /kg body weight.

2. Adjuvant composition

2.1 Nucleic acid adjuvant

The “adjuvant composition” according to the present invention contains a nucleic acid (nucleic acid adjuvant) composed of double-stranded RNA and a single-stranded oligodeoxynucleotide (single-stranded ODN) delivered to the endosome.

(1) “Double-stranded RNA”

Double-stranded RNA derived from microorganisms is known to activate natural immunity as a ligand for toll-like receptor 3 (TLR3). The same phenomenon is also seen in artificially synthesized double-stranded RNA (Matsumoto M., and T. Seya. 2008. TLR3: Interferon induction by double-stranded RNA including poly(I:C). Adv. Drug Del. Rev. 60: 805 -812.). TLR is a type 1 membrane protein that recognizes components derived from viruses or bacteria and initiates a biological defense response. TLR3 is one of the TLR family, and uses extracellular double-stranded RNA as a ligand to induce various cellular responses through TICAM-1. TLR3 is localized to endosomes in myeloid dendritic cells, and to the cell surface and endosomes in some epithelial cells and macrophages. However, in any cell, signals through TLR3 are transmitted from the endsomes, so double-stranded RNA is stored in the cell. It becomes necessary to introduce

Since double-stranded RNA derived from microorganisms does not act on human genes, research is also underway to use them as TLR3 ligands to control natural immunity and as vaccine adjuvants. The “double-stranded RNA” used in the present invention is an exogenous (e.g., virus or bacterial) double-stranded RNA that does not affect human endogenous genes and acts as a TLR3 ligand (with TLR3 activation ability). ), activates natural immunity.

The “double-stranded RNA” of the present invention can be synthesized, for example, by isolating dsRNA derived from an RNA virus and using this as a template. The target RNA virus is not particularly limited, and may be any of minus-strand single-stranded RNA viruses, plus-stranded single-stranded RNA viruses, and double-stranded RNA viruses. Specifically, measles virus, Sendai virus, RS virus, hepatitis C virus, polio virus, rota virus, etc. Preferred are minus-strand single-stranded RNA viruses such as measles virus, Sendai virus, and RS virus, and more preferably measles virus, Sendai virus, and RS virus vaccine strains.

DiRNA (defective interference RNA: SEQ ID NO: 1) derived from the Edmonston (ED) strain, a purified measles virus strain, has been traditionally included in vaccines (Shingai ey al., J Immunol. 2007; 179: 6123-6133). , safety for humans has been established. The inventors discovered that diRNA derived from this ED strain has an adjuvant function (Itoh et al., The Journal of Immunology, 2008; Matsumoto et al. Nat. Commun. 2015, as mentioned above), and produced Double-stranded RNA with a length of 59 to 140 bases is efficiently delivered to the endsome by linking it with a single-stranded ODN, which will be described later, and activates only TLR3 without activating the intracellular RNA sensors RIG-I and MDA5, leading to tumor regression. It was confirmed to be effective (WO2012/014945 and WO2016/088784). "Double-stranded RNA" based on diRNA derived from this ED strain specifically activates only TLR3 without activating other immune systems (e.g., MDA5/RIG-1) by capping the 5' end, so it is systemic. There was no risk of induction of inflammatory cytokines, making it excellent as a vaccine adjuvant. Therefore, a preferred form of "double-stranded RNA" used in the present invention includes double-stranded RNA based on diRNA derived from this ED strain, but double-stranded RNA derived from other viruses and bacteria can also be used as a TLR3 ligand, It can be used in the nucleic acid adjuvant of the present invention.

The length of the “double-stranded RNA” is not particularly limited, but for TLR3 activation ability, it is preferably about 50 base pairs or more, more preferably about 60 base pairs or more, about 90 base pairs or more, about 100 bases or more in length, and about 110 bases or more. is more preferred, and a length of at least about 120 bases is particularly preferred. The upper limit is not particularly limited, but for ease of synthesis, the length is preferably up to about 160 base pairs. Therefore, the length of the “double-stranded RNA” of the present invention is preferably about 50 to 160 base pairs, more preferably about 90 to 150 base pairs, about 100 to 150 base pairs, about 110 to 140 base pairs, and about 120 to 140 base pairs. This is particularly desirable.

The diRNA derived from ED strain has the nucleotide sequence shown in SEQ ID NO: 1 and forms a double-stranded RNA (diRNA) with a loop structure (Shingai et al., J Immunol. 2007 Nov 1; 179(9): 6123-6133 ). When designing “double-stranded RNA” based on diRNA derived from the ED strain, any part of the diRNA of the ED strain shown in SEQ ID NO: 1 may be used as the “double-stranded RNA,” but the AU-rich region is not effective in terms of synthesis efficiency. desirable. Such AU-rich regions include, for example, positions 17 to 78, 101 to 164, and 216 to 269 of SEQ ID NO: 1 as sense strand side sequences, but all are preferable regions in terms of synthesis efficiency. , and its function as a TLR3 ligand is not limited to these regions.

In the above-mentioned patent application (WO2012/014945), for diRNA of the ED strain shown in SEQ ID NO: 1, double-stranded RNA having the following base sequence was prepared and its activity was confirmed.

Base sequence from positions 1017 to 1074 (SEQ ID NO: 2),

Base sequence with three cytosines added to the 5' end of positions 1077 to 1152 (SEQ ID NO: 3)

Base sequence with three cytosines added to the 5' end of positions 1017 to 1152 (SEQ ID NO: 4)

Base sequence with three cytosines added to the 3' end of positions 1077 to 1152 (SEQ ID NO: 5)

Base sequence with three cytosines added to the 3' end of positions 1017 to 1152 (SEQ ID NO: 6)

Base sequence with three cytosines added to the 3' end of positions 1057 to 1152 (SEQ ID NO: 7)

Base sequence with three cytosines added to the 3' end of positions 1046 to 1152 (SEQ ID NO: 8)

Base sequence with three cytosines added to the 3' end of positions 1037 to 1152 (SEQ ID NO: 9)

Base sequence from positions 1017 to 1089 (SEQ ID NO: 10).

In addition, in the patent application (WO2016/088784), in the diRNA of the ED strain shown in SEQ ID NO: 1, a double-stranded RNA having a nucleotide sequence (SEQ ID NO: 11) from the 1st to 140th positions was prepared, and its activity was confirmed. did. Considering that diRNA forms a stem loop, it can be understood that the sequence shown in SEQ ID NO: 11 is a region complementary to the sequences shown in SEQ ID NOS: 2 to 9.

Therefore, as the “double-stranded RNA” of the present invention, a nucleic acid (double-stranded RNA) having a 30 to 160 base length continuous nucleotide sequence shown in SEQ ID NO: 1 can be used. In addition, in terms of synthetic efficiency, it contains at least 30 consecutive bases, preferably at least 40 bases, from positions 17 to 78, positions 101 to 164, and positions 216 to 269 of the base sequence shown in SEQ ID NO: 1. Nucleic acids (double-stranded RNA) with a total length of 100 to 160 bases (which may be included repeatedly), preferably 110 to 150 bases in length, and more preferably 120 to 140 bases in length, can be suitably used. . Additionally, “double-stranded RNA” may contain the partial sequence of SEQ ID NO: 1 repeated 2 to 4 times. For example, in addition to comprising the contiguous 120 base length of positions 17 to 136 of SEQ ID NO: 1 (SEQ ID NO: 42 (sense), SEQ ID NO: 43 (antisense)), the contiguous length of positions 17 to 56 of SEQ ID NO: 1 Those containing 40 bases in length repeated three times (SEQ ID NO: 44 (sense), SEQ ID NO: 45 (antisense)) can also be used.

A nucleic acid having a base sequence shown in any of SEQ ID NOs: 2 to 11 is an example, and among these, the inventors confirmed that the nucleic acid having a base sequence shown in SEQ ID NO: 11 and a partial sequence thereof has a tumor-reducing effect in vivo. .

Considering the function and structure of the "double-stranded RNA" of the present invention and diRNA derived from ED strain, it is not limited to nucleic acids (double-stranded RNA) having the nucleotide sequences shown in SEQ ID NOs. 2 to 11, and 100 or more consecutive bases thereof. , preferably 110 bases or more, more preferably 120 bases or more, double-stranded RNA can also be used. In addition, double-stranded RNA having a nucleotide sequence having 80% sequence identity, preferably 85%, more preferably 90%, and even more preferably 95% sequence identity with the above sequence, as long as it has TLR3 activation ability. , can be used as “double-stranded RNA” of the present invention. In addition, when the “double-stranded RNA” contains repeated partial sequences, the sequence identity is a value obtained by comparing the partial sequence with the sequence corresponding to the partial sequence.

In addition, in the base sequence shown in SEQ ID NOs: 2 to 11, 100 or more consecutive bases, preferably 110 or more bases, more preferably 120 or more bases, 1 to 10 bases, preferably 1 to 6 bases. , more preferably double-stranded RNA having a base sequence in which 1 to 3 bases are deleted, substituted, inserted, or added can also be used as the “double-stranded RNA” of the present invention as long as it has TLR3 activation ability.

As described in Example 2 of WO2012/014945, TLR3 activation ability was determined by constructing cells transfected with a human TLR3 expression plasmid together with a reporter plasmid containing an IFN-β promoter, and adding double-stranded RNA to the culture medium. This can be easily confirmed by adding and observing the increase in reporter activity. Therefore, it is not limited to the "double-stranded RNA" shown in SEQ ID NO: 2 to 11, and those skilled in the art will know the nucleotide sequence of dsRNA derived from a virus or bacteria as shown in SEQ ID NO: 1, the description in WO2012/014945 and WO2016/088784, and From the technical knowledge in the field, other “double-stranded RNA” used in the present invention can be easily obtained.

(2) Single-chain oligodeoxynucleotide (single-chain ODN)

TLR9 recognizes unmethylated CpG motifs in viral or bacterial DNA. Signaling through TLR9 is accomplished with high efficiency by short synthetic oligodeoxynucleotides (ODNs). This ODN contains a CpG motif unique to the genes of bacteria and viruses, and is well known in the field as “CpG ODN.” Since CpG ODN is delivered to the endsomes of dendritic cells and functions as a TLR9 ligand, it is known to be useful as a strong vaccine adjuvant or antibody production enhancer. On the other hand, it strongly induces MyD88-dependent cytokines (especially IFN-γ) and causes a Th2 response. CpG ODN is classified into three types, type A is an activator of NK cells that strongly induces IFNα, type B is an activator of B cells that weakly induces IFNα, and type C is an activator of type B and type A. It has both functions. The “single-chain ODN” of the present invention is preferably a GpC type in which the cytokine-inducing activity of CpG has been removed, and is preferably based on a B-type or C-type GpC ODN.

In the above-mentioned patent application (WO2012/014945), in the CpG ODN, the ODN obtained by converting CpG to GpC (GpC ODN) or the ODN converted to TpC or CpC is also delivered to the endsome. confirmed by them. In addition, the present inventors confirmed that nucleic acids containing a partial sequence of the ODN with a length of 5 bases or more are also delivered to the endsome.

ODN, obtained by converting CpG into GpC, TcP, or CPC, is delivered to the endosomes of dendritic cells, but does not have TLR9 agonist activity, so it does not induce unnecessary immune responses. That is, the “single-chain ODN” used in the present invention is a single-chain ODN delivered to the endsome that does not have TLR9 agonist activity.

CpG ODN is a TLR9 agonist, and GpC ODN is commercially available as its control (since it does not have TLR9 agonist activity), and these ODNs can be suitably used in the present invention. Below, the CpG ODN (B type or C type) and the GpC ODN (control) sold from Invivogen and their sequences are described (http://www.invivogen.com/tlr9-agonist).

The "single-stranded ODN" of the present invention includes ODNs having a base sequence in which CpG dinucleotides are replaced with GpC, TpC or CpC dinucleotides in CpG ODNs, or partial sequences thereof having an endosome delivery function.

Specific examples of such “single-chain ODN” include ODNs having the base sequences shown in SEQ ID NOs: 19 to 39, or nucleic acids containing partial sequences having a length of 5 bases or more.

In addition, in the sequences shown in SEQ ID NOs: 19 to 39, ODNs having 1 to 3 base sequences, preferably 1 or 2 bases, and more preferably 1 base sequences having deletions, substitutions, insertions or additions, are also TLR9 As long as it does not have a nist activity and is delivered to the endsome, it can be used as a “single-chain ODN” of the present invention.

The "single-strand ODN" of the present invention is preferably 15 bases or more in length, and more preferably 15 to 28 bases in length, from the viewpoint of the delivery function to endosomes.

The ability to deliver to endosomes is as described in Example 5 of WO2012/014945, by producing cells transfected with human TLR3 together with a reporter plasmid containing an IFN-β promoter, and injecting double-stranded cells into the culture medium. This can be easily confirmed by adding a nucleic acid linked to RNA with a single-stranded ODN and comparing the reporter activity with the control. Therefore, in addition to the single-chain ODN having the nucleotide sequence shown in SEQ ID NOs: 19 to 39, those skilled in the art will know from the nucleotide sequences of commercially available CpG ODN and GpC ODN (control) and the descriptions of WO2012/014945 and WO2016/088784 that the present invention Other “single chain ODN” used in can be easily obtained.

The single-stranded nucleotides constituting the “GpC ODN” of the present invention are preferably modified from the viewpoint of stability (nuclease resistance). Examples of such modification include phosphorothioate modification. By being modified with phosphorothioate, single-stranded ODN is not degraded by nucleases and can efficiently deliver nucleic acids to endosomes.

(3) Synthesis of adjuvant nucleic acid

The adjuvant nucleic acid of the present invention is composed of the above-described “double-stranded RNA” and “single-stranded ODN.” The order in which “double-stranded RNA” and “single-stranded ODN” are linked or the linking style are not particularly limited, and even if the 5’ side of “double-stranded RNA” is linked to the 3’ side of “single-stranded ODN”, “single-stranded RNA” The 3' side of the "double-stranded RNA" may be bound to the 5' side of the "single-stranded ODN," but it is preferable that the 5' side of the "double-stranded RNA" be bound to the 3' side of the "single-stranded ODN." It is more preferable that the 3' end of the "single-stranded ODN" is linked to the 5' end of the sense strand of the "double-stranded RNA".

“Double-stranded RNA” and “single-stranded ODN” may be linked directly or through an appropriate linker. The length (number of bases) of the linker sequence is not limited. In addition to the linker sequence, another nucleic acid may be linked to one or both ends, but in this case, it is preferable to link the other nucleic acid to the 3' side of the RNA.

It is preferable that each single-stranded nucleic acid forming the nucleic acid of the present invention does not have a phosphate group bound to any terminal. If a phosphate group remains at the 5' end, large-scale administration in vivo activates the RIG-I pathway in the cytoplasm, leading to the production of large amounts of cytokines, which can cause side effects (Robinson RA, DeVita VT, Levy) HB, Baron S, Hubbard SP, Levine AS. A phase I-II trial of multiple-dose polyriboinosic-polyribocytidylic acid in patients with leukemia or solid tumors. J Natl Cancer Inst. 1976 Sep; 57(3): 599-602) . A triphosphate is added to the 5' end of the RNA chain synthesized by in vitro transcription, but since the nucleic acid of the present invention can be produced by chemical synthesis, no phosphate group is attached to either the 5' or 3' end. It can be synthesized as a nucleic acid.

When the 3' end of the "single-stranded ODN" is directly covalently bonded to the 5' end of the sense strand of the "double-stranded RNA", the nucleic acid adjuvant of the present invention can be used for single-stranded nucleic acid A ("single-stranded ODN" and " It can be synthesized as a nucleic acid containing a chimeric nucleic acid of a “double-stranded RNA” sense strand) and a single-stranded nucleic acid B (a “double-stranded RNA” antisense strand).

As an example, WO2016/088784 describes a nucleic acid consisting of a sense strand (single-stranded nucleic acid A) shown in SEQ ID NO: 40 and an antisense strand (single-stranded nucleic acid B) shown in SEQ ID NO: 41, and a method for synthesizing the same. In this example, “double-stranded RNA” and “single-stranded ODN” are directly linked, but a linker may be included between them as long as it does not have a negative effect on the stability and adjuvant activity of the nucleic acid. In this specification, the nucleic acid adjuvant composed of the sense strand shown in SEQ ID NO: 40 and the antisense strand shown in SEQ ID NO: 41 may be referred to as "ARNAX".

The nucleic acid of the present invention is not limited to the sense strand and the antisense strand, and has a sense sequence identity of 80%, preferably 85%, more preferably 90%, and even more preferably 95%, respectively, with the above sequence. Nucleic acids composed of a strand and an antisense strand can also be used as nucleic acids of the present invention as long as they have adjuvant activity. In addition, as described above, when "double-stranded RNA" repeatedly contains a specific partial sequence, the sequence identity is a value obtained by comparing the partial sequence with the sequence corresponding to the partial sequence for the repeated portion. do.

The adjuvant activity of the nucleic acid is, for example, primarily transfected with a human TLR3 expression plasmid together with a reporter plasmid containing an IFN-β promoter, according to the examples described in WO2012/014945 and WO2016/088784. It can be confirmed by producing it, adding a nucleic acid adjuvant to the culture medium, and observing the increase in reporter activity, and more precisely, by looking at the tumor regression effect in vivo.

Nucleic acids containing the above-described sense strand and antisense strand can be synthesized, for example, by synthesizing partial sequences and sequentially ligating them according to the method described in WO2016/088784. Of course, the method is not limited to the above, and the synthesis method may be optimized appropriately according to conventional methods depending on the length and structure of the double-stranded RNA and single-stranded ODN used.

In the microenvironment of a tumor, a state of immune control occurs not only by tumor cells but also by myeloid cells that have infiltrated the tumor. Tumor-associated macrophages (TAMs) strongly support tumor proliferation, maintenance, and invasion, contribute to the formation of a healthy microenvironment for tumors, and myeloid-derived suppressor cells (MDSCs). )) inhibits the activity of antigen-specific T cells. When poly(I:C) treatment is performed on mouse transplant cancers in which these suppressive macrophages infiltrate the tumor, the tumor regresses (Shime et al. Proc. Natl. Acad. Sci. U.S.A. 109(6): 2066-2071.2012) . The factor for tumor regression is hemorrhagic necrosis of the tumor due to TLR3-TICAM-1-dependent production of TNF-α from TAM in response to poly(I:C), but at the same time, macrophages within the tumor are effectors from the suppressive M2 type. It is converted to the M1 type, and epigenetic changes are induced in the TLR3 signal. It is believed that administration of the adjuvant composition of the present invention, which is a TLR3-specific adjuvant, can inhibit tumor growth by converting cancer suppressor cells within a tumor into a cancer-attacking type and controlling the microenvironment within the tumor.

The nucleic acid adjuvant of the present invention 1) is efficiently introduced into dendritic cells, 2) activates only TLR3 and does not activate MDA5/RIG-1, and 3) does not induce systemic inflammatory cytokine/type I IFN production. , 4) activates dendritic cells, induces NK cells and CTLs, 5) exhibits strong anti-tumor activity, 6) can inhibit tumor proliferation by controlling the microenvironment within the tumor, 7) according to GMP standards It has the characteristic of being capable of chemical synthesis. Therefore, it is very useful as an adjuvant in cancer immunotherapy.

(4) Formulation

The “adjuvant composition” of the present invention may contain a pharmacologically acceptable carrier or additive. Such carriers and additives include, for example, surfactants, excipients, colorants, flavoring agents, preservatives, antioxidants, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity promoters, coagulants, etc. However, it is not limited to these, and other commonly used carriers may be appropriately used. Specific examples of these are as described in “Immune check point inhibitors.”

The route of administration of the “adjuvant composition” of the present invention is not particularly limited, but parenteral administration is preferred, and specific examples include injection administration, nasal administration, transpulmonary administration, and transdermal administration. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The administration method can be appropriately selected depending on the patient's age and symptoms.

The dosage of the “adjuvant composition” of the present invention is appropriately determined depending on the purpose of use, route of administration, etc. When administering to humans, for example, the dosage can be selected in the range of 0.00001 mg to 10 mg per kg of body weight for one administration. Alternatively, the dosage may be selected in the range, for example, from 1 to 100 mg/body per patient. However, the dosage of the “adjuvant composition” of the present invention is not limited to the above dosage.

2.2 Antigen Molecules

In the present invention, the adjuvant composition may contain an antigen molecule. Since endogenous antigens exist in the bodies of cancer patients and bacterial infections, an immune effect is obtained with only adjuvant nucleic acids. Above all, in cases where a sufficient effect cannot be obtained with the adjuvant nucleic acid alone, it is preferable to administer the antigen molecule together with the adjuvant, depending on the disease being treated. Antigen molecules include viral antigens, bacterial antigens, cancer antigens, and antigenic components thereof.

Viral antigens include, for example, adenovirus, retrovirus, picornavirus, herpesvirus, rotavirus, hantavirus, coronavirus, togavirus, flavivirus, rhabdovirus, paramyxovirus, and orthomyxovirus. , Beniya virus, Arena virus, Reo virus, Papilloma virus, Parvovirus, Pox virus, Hepadna virus, Spongiovirus, HIV, CMV, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Influenza virus. , measles virus, polio virus, smallpox virus, rubella virus, herpes simplex virus, varicella zoster virus, Epstein Barr virus, Japanese encephalitis virus, rabies virus, influenza virus, or a combination thereof.

Examples of bacterial antigens include bacterial antigens such as Bacillus, Escherichia, Listeria, Neisseria, Nocardia, Salmonella, Staphylococcus, and Streptococcus, or combinations thereof. .

Cancer antigens include, for example, leukemia, lymphoma, glioblastoma, glioblastoma, melanoma, breast cancer, lung cancer, head and neck cancer, digestive system tumor, stomach cancer, colon cancer, liver cancer, pancreatic cancer, uterine cancer, ovarian cancer, vaginal cancer, testicular cancer, and prostate cancer. Cancer, penile cancer, bone tumor, vascular tumor, esophageal cancer, rectal cancer, colon cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, laryngeal cancer, bronchial cancer, bladder cancer, kidney cancer, brain tumor, thyroid cancer, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, etc. Antigens or combinations thereof may be mentioned.

The adjuvant composition of the present invention alone has a therapeutic effect for cancer or infectious diseases, and the present invention also provides a medicine for treating cancer or infectious diseases containing the adjuvant composition.

3. Medicines to treat cancer or infections

The present invention provides a medicine for treating cancer or infectious diseases comprising a combination of the above-described immune checkpoint inhibitor and adjuvant composition.

The medicine of the present invention refers to a medicine in which an immune checkpoint inhibitor containing an anti-PD-1 antibody or an anti-PD-L1 antibody and an adjuvant composition are combined (administered in combination) for simultaneous, separate or sequential administration. do.

The immune check point inhibitor and the adjuvant composition may be provided separately, or may be provided as a kit consisting of the immune check point inhibitor and the adjuvant composition.

Diseases that can be treated by the medicine of the present invention include diseases that can be targeted by immune checkpoint inhibitors (cancer, infectious diseases, etc.). Cancers include, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, Vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, parenchymal sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Chronic leukemia, acute leukemia, pediatric solid cancer, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal pelvis carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, tumor renal vasculature, spinal tumor, brainstem tumor, pituitary adenoma, Examples include Kaposi's sarcoma, squamous cell carcinoma, squamous cell carcinoma, T-cell lymphoma, and environmentally induced tumors. In particular, it can be suitably applied to metastatic cancer expressing PD-L1.

Examples of infectious diseases include HIV infection (AIDS), hepatitis, herpes, malaria, leishmaniasis, influenza, dysentery, pneumonia, tuberculosis, sepsis, and the like. In particular, it can be suitably applied to HIV infection, which causes severe immune deficiency.

As described above, while anti-PD-1 antibodies and anti-PD-L1 antibodies act in the effector phase of the immune defense mechanism, the nucleic acid adjuvant of the present invention is delivered to the endosomes of dendritic cells and activates TLR3, It is thought to act in the priming phase, such as inducing NK cells and CTL. Therefore, by using the adjuvant nucleic acid of the present invention in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody, the immune defense mechanism can be activated through both the priming phase and the effector phase (see Figure 5).

Due to the above-mentioned mechanism of action, the drug of the present invention uses the adjuvant composition and anti-PD-1 antibody and anti-PD-L1 antibody in combination, thereby preventing the use of anti-PD-1 antibody or anti-PD-L1 antibody alone. It is also effective against cancer and induces more powerful CTL-dependent tumor regression. By using the adjuvant composition and anti-PD-1 antibody or anti-PD-L1 antibody in combination, the antitumor effect is synergistically increased compared to administering each individually.

In addition, by using it in combination with an adjuvant composition, effects such as antibody production and NK cell activation and improvement of the tumor microenvironment that cannot be achieved with anti-PD-1 antibodies or PD-L1 antibodies alone are also exhibited. The medicine of the present invention has high safety and can be safely administered even to elderly people, expanding the range of treatment targets using anti-PD-1 antibodies or anti-PD-L1 antibodies, and increasing its effectiveness.

Example

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Example 1: Induction of CTL by administration of ARNAX and antigen

1. Materials and Methods

Wild type (C57BL/6) and knockout ♂ mice (MAVS KO, MyD88 KO, TLR3 KO, TICAM-1 KO, IRF3 KO, IRF3/7D KO) on B57BL/6 background, 8 to 11 weeks old, were used in the experiments. PBS, OVA (100 μg), or OVA (100 μg) + ARNAX (60 μg) were administered subcutaneously to mice on day 0 and day 7. On day 11, the spleen and subgenual lymph nodes were collected, and OVA-specific CD8 ⁺ T cells among CD8 ⁺ T cells were collected. The ratio was measured with OVA-tetramer (manufactured by MBL). Tetramer assay was performed according to the attached document. Specifically, spleen and lymph node cells (1x10 ⁷ /ml) were stained with 50-fold diluted H2-Kb OVA tetramer (PE) for 20 minutes, washed, and then stained with anti-CD3em Ab (APC), anti-CD8am Ab ( FITC) was stained, and the ratio of OVA-specific CD8 + T cells among CD8 + T cells was analyzed using FACS Aria (BD). Additionally, splenocytes (1x10 ⁷ /ml) were stimulated with 100 nM of OVA-specific peptide (SL8: SIINFEKL) or control WT1 peptide (Db126: RMFPNAPYL) for 3 days, and IFN-γ in the culture supernatant was assayed using a cytokine bead assay (Cytokine bead assay). It was measured by bead assay (CBA; BD).

2. Results

In wild-type mice, OVA-specific CTL were not induced in the spleen and subgenual lymph nodes by administration of PBS or OVA alone, but were induced by administration of OVA+ARNAX. Knockout mice of MAVS, an adapter molecule downstream of RIG-I-like receptor (RLR), and MyD88 knockout mice, an adapter molecule of TLRs other than TLR3, showed the same results as wild-type mice. In mice knocking out TLR3, TICAM-1, an adapter molecule of TLR3, and IRF3 and IRF7, transcription factors essential for type I IFN induction, OVA-specific CTL were not completely induced by OVA+ARNAX (Figure 1a, c). In addition, in splenocytes of wild-type mice and MAVS knockout mice, a large amount of IFN-γ was produced upon stimulation with OVA-specific peptide, but no complete production of IFN-γ was observed in TLR3, TICAM-1, IRF3, and IRF3/7 knockout mice. (b in Figure 1).

3. Considerations

It became clear that antigen-specific CTL is not induced by antigen alone, but is induced by antigen + ARNAX administration. From the results of knockout mice, it was revealed that antigen-specific CTL induction by ARNAX depends on the TLR3-TICAM-1-IRF3 pathway, and does not involve the RLR-MAVS pathway or the TLR-MyD88 pathway other than TLR3. The priming adjuvant activity of ARNAX can be defined as the TLR3-TICAM-1 signal.

Example 2: Comparison of the cytokine production effect and safety of ARNAX by administration route

1. Materials and Methods

8-week-old wild-type (C57BL/6) ♀ mice were administered saline, poly(I:C) (150 μg), and ARNAX (150 μg) subcutaneously, or intravenously, saline, poly(I:C) (50 μg), and ARNAX (50 μg) was administered, and the amount of cytokines in the blood was measured 3 and 6 hours later (Figure 2). IL-6, TNF-α, and IL-12p40 were measured by CBA, and IFN-βIP-10 was measured by ELISA.

2. Results

When ARNAX was administered subcutaneously, the inflammatory cytokines IL-6 and TNF-α in the blood were lower than when administered with poly(I:C), and IFN-γ was also very low. Meanwhile, IL-12p40, a Th1-type cytokine, is induced to be produced more than poly(I:C), and IP-10, a chemokine that recruits NK cells, NKT cells, and T cells, is induced equally to poly(I:C). It has been done. Upon intravenous administration, poly(I:C) strongly induced TNF-α, IL-6, and IFN-β, but barely induced ARNAX.

3. Considerations

Regardless of the route of administration, ARNAX can be said to be a non-inflammatory adjuvant with reduced side effects seen with poly(I:C) in that it rarely induces the production of systemic inflammatory cytokines or type I IFN. On the other hand, IL-12, a Th1 type cytokine, is produced and induced to a greater extent than poly(I:C). Antigen-specific CTL induction requires cytokines such as IL-12 or type I IFN as a third signal, but CTL induced by IL-12 is more dependent on PD-1 within the tumor than CTL induced by type I IFN. There are reports of lower expression and longer survival, and the IL-12 ^high /IFN-β ^low cytokine profile of ARNAX was suitable for the induction of functional antigen-specific CD8 ⁺ T cells within tumors.

Example 3: Antitumor effect by administration of ARNAX and antigen (thymoma)

1. Materials and Methods

EG7 ( ^2x106 /200μl PBS), an OVA-expressing tumor (thymoma), was subcutaneously transplanted into the back and lower back of C67BL/6 mice (7 weeks old, ♀), and on day 7, PBS (n=5) or OVA (100μg)+ARNAX ( 60 μg) (n = 5) was administered subcutaneously, and tumor growth was measured over time (Figure 3c). Tumors were collected on Day 14, and the proportion of CD8 ⁺ T cells in the tumor, OVA-specific CD8 ⁺ T cells among CD8 ⁺ T cells, and CD11c-positive CD8 ⁺ T cells were measured by flow cytometry (Figure 3d) ). In addition, tumor tissue sections were prepared, immunostained with anti-CD8α antibody, and the infiltration of CD8 ⁺ T cells within the tumor was quantified by observing 20 fields of view in the PBS group and 16 fields of view in the OVA+ARNAX group with a confocal microscope (Figure 3f) ). In addition, RNA was purified from tumor tissue using Trizol, and gene expression of Gzmb, Ifng, Perf1, Cxcl9, Cxcl10, and Cxcl11 was measured by quantitative PCR (Figure 3e).

2. Results

In OVA+ARNAX treatment, CD8 ⁺ T cells were highly infiltrated within the EG7 tumor (Figure 3d, f). The proportion of OVA-specific CTL among CD8 ⁺ T cells was high, and the activation marker CD11c was also expressed, so it is thought that tumor-reactive CTL of effector memory were induced. As a result, tumor regression was induced (Figure 3c). Within the tumor, genes such as granzyme (Gzmb), perforin (Prf1), IFN-γ (Ifng), Cxcl9, 10, and 11 were expressed at elevated levels in OVA+ARNAX (Figure 3) e).

3. Considerations

It is believed that administration of ARNAX+cancer antigen can efficiently induce tumor-specific CTL to infiltrate the tumor and induce tumor regression. Within the tumor, the expression of cell injury-related genes such as granzyme, perforin, and IFN-γ and chemokine genes that induce CTL such as Cxcl9, 10, and 11 are elevated, and ARNAX can establish a Th1-type immune response within the tumor. It is an adjuvant that can be used.

Example 4: Combined administration of ARNAX and PD-L1 antibody (thymoma)

1. Materials and Methods

PD-L1 high-expressing EG7 (Kataoka et al., Nature 534: 402, 2016) (2x10 ⁶ /200 μl PBS) was subcutaneously implanted on the back of C67BL/6 mice (7 weeks old, ♀), and on day 7, PBS (isolated) type (isotype Ab group) (n = 4), PBS (anti-PD-L1 Ab group) (n = 4), OVA (100 μg) + ARNAX (60 μg) (isotype Ab group) (n = 6), OVA (100 μg) + ARNAX (60 μg) (anti-PD-L1 Ab group) (n = 6) was administered subcutaneously, and isotype Ab or anti-PD-L1 Ab (200 μg/head) was administered on days 7, 9, and 11. Tumor proliferation was measured over time by intravenous administration (Figure 4c). The spleen and tumor were collected on Day 13, and the percentage of CD8 ⁺ T cells in the spleen and tumor, the percentage of OVA-specific CD8 ⁺ T cells, CD11c-positive CD8 ^{+ T} cells, and PD-1-positive CD8 ⁺ T cells among CD8 + T cells. was measured by flow cytometry (Figure 4d). Additionally, RNA was purified from tumor tissue using Trizol, and gene expression of Gzmb, Ifng, Perf1, Cxcl9, Cxcl10, and Cxcl11 was measured by quantitative PCR (Figure 4e).

2. Results

In EG7 with high PD-L1 expression, tumor growth was not suppressed by PD-L1 antibody treatment alone, but tumor regression was induced by OVA+ARNAX. Additionally, the tumor was regressed more effectively when used in combination with the PD-L1 antibody (Figure 4c). In OVA+ARNAX, CD8+T cells were infiltrated into the tumor, and when anti-PD-L1 antibody was used together, more T cells were infiltrated. The proportion of OVA-specific CD8 + T cells in the tumor was elevated for both OVA + ARNAX and OVA + ARNAX + anti-PD-L1 Ab (Figure 4d). Meanwhile, in the spleen, the proliferation of antigen-specific CD8 + T cells was significantly increased with the combined use of PD-L1 antibody, and the proportion of activation markers CD11c and PD-1 positive CD8 + T cells was also high, indicating tumor reactivity of effector memory. It was found that CTL was induced. In addition, gene expression of Gzmb, Ifng, Perf1, and Cxcl9 in the tumor was also increased by the combined use of ARNAX+OVA and anti-PD-L1 antibody (Figure 4e).

3. Considerations

In EG7 tumors with high PD-L1 expression that are resistant to PD-L1 antibody treatment, tumor growth is suppressed to some extent with OVA+ARNAX, but tumor regression can be effectively induced by combined use of anti-PD-L1 antibodies. , it is believed that PD-1/PD-L1 resistance can be eliminated by combined treatment.

It is believed that by blocking the PD-1 pathway, many tumor-specific CTLs are induced during priming and infiltrate into the tumor, and cancer regression is effectively induced by the release of functional inhibition on the effector phase, and anti-PD-1/PD-L1 Combination with antibodies is useful for enhancing the action of ARNAX (see Figure 5).

Example 5: Combined administration of ARNAX and PD-L1 antibody (melanoma)

1. Materials and Methods

MO5 (2x10 ⁶ /200μl PBS), an OVA-expressing B16 melanoma, was subcutaneously implanted on the back of C67BL/6 mice (7 weeks old, ♀), and on day 10, PBS (isotype Ab group) (n = 5), PBS ( anti-PD-L1 Ab group) (n = 4), OVA (100 μg) + ARNAX (60 μg) (isotype Ab group) (n = 4), OVA (100 μg) + ARNAX (60 μg) (anti-PD-L1 Ab group) (n=4) was administered subcutaneously, isotype Ab or anti-PD-L1 Ab (200 μg/head) was administered intravenously on days 10, 12, 14, and 16, and tumor growth was measured over time. (c in Figure 6). Spleen and tumor were harvested on Day 17, percentage of CD8 ⁺ T cells in spleen and tumor, percentage of OVA-specific CD8 ^{+ T} cells and CD11c positive CD8 ⁺ T cells, percentage of PD-1 positive CD8 ⁺ T cells among CD8 + T cells. was measured by flow cytometry (Figure 6d). In addition, RNA was purified from tumor tissue using Trizol, and gene expression of granzyme (Gzmb), perforin (Prf1), IFN-γ (Ifng), and Cxcl10 was measured by quantitative PCR (Figure 6e) . Additionally, the proportion of tumor-infiltrating immune cells was measured for lymphocytes, dendritic cells, and CD11b-positive myeloid cells (FIG. 6f).

2. Results

In PD-L1 expressing MO5, OVA+ARNAX induced tumor regression to the same degree as PD-L1 antibody treatment alone. Additionally, OVA+ARNAX regressed tumors more effectively when used in combination with PD-L1 antibody (Figure 6c). In OVA+ARNAX, CD8+T cells were infiltrated into the tumor, and when anti-PD-L1 antibody was used together, more T cells were infiltrated. The proportion of OVA-specific CD8 + T cells in the tumor was elevated for both OVA + ARNAX and OVA + ARNAX + anti-PD-L1 Ab (Figure 6d). Meanwhile, in the spleen, the proliferation of antigen-specific CD8 + T cells is significantly increased with the combined use of PD-L1 antibody, and the proportion of activation markers CD11c and PD-1 positive CD8 ⁺ T cells is also high, indicating tumor reactivity of effector memory. It was confirmed that CTL was induced. In addition, gene expression of Gzmb, Ifng, Perf1, and Cxcl9 in the tumor was also increased by the combined use of ARNAX+OVA and anti-PD-L1 antibody (Figure 6e).

3. Considerations

For MO5 tumors, ARNAX+cancer antigen treatment inhibits tumor proliferation, but combined use with PD-L1 antibody can more effectively induce tumor regression. From this, it is thought that PD-1/PD-L1 immune checkpoint inhibitors can exert a high anti-tumor effect also for melanoma by combined treatment with ARNAX + cancer antigen.

Example 6: Structure of ARNAX and ability to activate IFNβ through TLR3

1. Materials and Methods

The following four types of double-stranded RNA were synthesized.

ARNAX140: double-stranded RNA having a base sequence from positions 1 to 140 of SEQ ID NO: 1

ARNAX120#1: double-stranded RNA having a nucleotide sequence from positions 17 to 136 of SEQ ID NO: 1

ARNAX120#2: Double-stranded RNA having a sequence of nucleotides 17 to 56 of SEQ ID NO: 1 linked three times

ARNAX130: double-stranded RNA having a base sequence from positions 1 to 130 of SEQ ID NO: 1

Positions 17 to 56 of SEQ ID NO: 1 are AU-rich regions (Figure 7).

HEK293 cells were transfected with control plasmid (pEF/BOS) or human TLR3 expression plasmid together with the IFN-β promoter reporter plasmid, and after 24 h, culture medium, poly(I:C), ARNAX140, or ARNAX120#2 were added at 2, 5, It was added at a concentration of 10 μg/ml. After 6 hours, luciferase activity in cells was measured. The luciferase activity when medium was added to pEF/BOS-expressing cells as a negative control was set as 1 and expressed as a fold (Figure 8).

The same experiment was performed by adding poly(I:C), ARNAX140, ARNAX130, or ARNAX120#1 at concentrations of 2, 5, and 10 μg/ml (FIG. 9).

2. Results

The ability of ARNAX120 to activate the IFN-β promoter through TLR3 is 7 to 8 percent that of ARNAX140, and no difference in TLR3 activation ability was confirmed between ARNAX120#1 and ARNAX120#2, which have different RNA sequences. It was confirmed that ARNAX130 has the ability to activate the IFN-β promoter through TLR3 equivalent to that of ARNAX140.

Example 7: CTL inducing activity of ARNAX120

1. Materials and Methods

According to Example 1, using 8-week-old wild-type (C57BL/6) female mice, PBS, OVA (100 μg), OVA (100 μg) + poly(I:C) (50 μg), OVA (100 μg) + ARNAX140 ( The ratio of OVA-specific CD8 ⁺ T cells in the spleen and subgenual lymph nodes was measured after subcutaneous administration of 10 μg), OVA (100 μg) + ARNAX140 (30 μg), and OVA (100 μg) + ARNAX140 (60 μg).

Similarly, using 8-week-old wild-type (C57BL/6) magnetic mice, PBS, OVA (100 μg), OVA (100 μg) + poly(I:C) (50 μg), OVA (100 μg) + ARNAX120#2 (10 μg) ), the ratio of OVA-specific CD8 ⁺ T cells in the spleen was measured by subcutaneous administration of OVA (100 μg) + ARNAX120#2 (30 μg). Additionally, splenocytes ( ^1x107 /ml) were stimulated with 100nM of OVA-specific peptide (SL8: SIINFEKL) or control WT1 peptide (Db126: RMFPNAPYL) for 3 days, and IFN-γ in the culture supernatant was measured by ELISA.

2. Results

Although OVA-specific CTL was not induced by administration of PBS or OVA alone, CTL was induced by administration of OVA+poly(I:C), OVA+ARNAX140, and OVA+ARNAX120#2, confirming cross-priming activity in vivo. (Figure 10, Figure 11). As a result of varying the dosage of ARNAX140, it was found that 10 μg/head had sufficient CTL inducing activity (FIG. 10). It was confirmed that ARNAX120 has a CTL-inducing activity (cross-priming activity) equivalent to that of ARNAX140, and that it exhibits sufficient CTL-inducing activity at a dose of 10 μg/head (Figure 11).

Example 8: Anti-tumor activity of ARNAX120

1. Materials and Methods

According to Example 3, EG7 (2x10 ⁶ /200μl PBS) was implanted subcutaneously on the back of C67BL/6 mice (7 weeks old, ♀), and on day 7, PBS (n = 5) and ARNAX120#1 (10μg) (n) =4) or OVA (100 μg)+ARNAX120#1 (10 μg) (n=5) was administered subcutaneously, and tumor growth was measured over time.

2. Results

Although 10 μg of ARNAX120#1 was administered alone, inhibition of EG7 tumor growth was observed, simultaneous administration with OVA antigen significantly regressed the tumor ( Fig. 12 ). From the above results, it was confirmed that ARNAX can sufficiently exert its effect if it is 120 bases long, and that its sequence only needs to contain at least 40 consecutive bases of the sequence shown in SEQ ID NO: 1. Since shorter nucleic acids are preferable in terms of production efficiency and cost, ARNAX120 and ARNAX130 can be useful adjuvant candidates from a practical standpoint.

According to the present invention, the effect or main efficiency of PD-1/PD-L1 antibody therapy can be improved. Therefore, the present invention is useful in medical fields where immunotherapy using immune checkpoint inhibitors (PD-1/PD-L1 antibodies) is expected, such as cancer and infectious diseases.

All publications, patents, and patent applications cited in this specification are hereby incorporated into this specification by reference.

SEQ ID NO: 12: Synthetic DNA (ODN M362)
SEQ ID NO: 13: Synthetic DNA (ODN2006)
SEQ ID NO: 14: Synthetic DNA (ODN1668)
SEQ ID NO: 15: Synthetic DNA (ODN1826)
SEQ ID NO: 16: Synthetic DNA (ODN2007)
SEQ ID NO: 17: Synthetic DNA (ODN BW006)
SEQ ID NO: 18: Synthetic DNA (ODN2395)
SEQ ID NO: 19: Synthetic DNA (ODN M362 control)
SEQ ID NO: 20: Synthetic DNA (ODN M362 Control_TpC Substituent)
SEQ ID NO: 21: Synthetic DNA (ODN M362 Control_CpC Substituent)
SEQ ID NO: 22: Synthetic DNA (ODN2006 control)
SEQ ID NO: 23: Synthetic DNA (ODN2006 Control_TpC Substituent)
SEQ ID NO: 24: Synthetic DNA (ODN2006 Control_CpC Substituent)
SEQ ID NO: 25: Synthetic DNA (ODN2007 control)
SEQ ID NO: 26: Synthetic DNA (ODN2007 Control_TpC Substituent)
SEQ ID NO: 27: Synthetic DNA (ODN2007 Control_CpC Substituent)
SEQ ID NO: 28: Synthetic DNA (ODN BW006 control)
SEQ ID NO: 29: Synthetic DNA (ODN BW006 Control_TpC Substituent)
SEQ ID NO: 30: Synthetic DNA (ODN BW006 Control_CpC Substituent)
SEQ ID NO: 31: Synthetic DNA (ODN2395 control)
SEQ ID NO: 32: Synthetic DNA (ODN2395 Control_TpC Substituent)
SEQ ID NO: 33: Synthetic DNA (ODN2395 Control_CpC Substituent)
SEQ ID NO: 34: Synthetic DNA (ODN1668 control)
SEQ ID NO: 35: Synthetic DNA (ODN1668 Control_TpC Substituent)
SEQ ID NO: 36: Synthetic DNA (ODN1668 Control_CpC Substituent)
SEQ ID NO: 37: Synthetic DNA (ODN1826 control)
SEQ ID NO: 38: Synthetic DNA (ODN1826 Control_TpC Substituent)
SEQ ID NO: 39: Synthetic DNA (ODN1826 Control_CpC Substituent)
SEQ ID NO: 40: DNA/RNA chimeric molecule for sense strand (ARNAX140)
SEQ ID NO: 41: Synthetic RNA for antisense strand (ARNAX140)
SEQ ID NO: 42: DNA/RNA chimeric molecule for sense strand (ARNAX120#1)
SEQ ID NO: 43: Synthetic RNA for antisense strand (ARNAX120#1)
SEQ ID NO: 44: DNA/RNA chimeric molecule for sense strand (ARNAX120#2)
SEQ ID NO: 45: Synthetic RNA for antisense strand (ARNAX120#2)
SEQ ID NO: 46: DNA/RNA chimeric molecule for sense strand (ARNAX130)
SEQ ID NO: 47: Synthetic RNA for antisense strand (ARNAX130)

SEQUENCE LISTING <110> National University Corporation Hokkaido University <120> Adjuvant for Cancer Immunotherapy <130> PHU-9004WO <150> JP2016-147657 <151> 2016-07-27 <150> JP2017-079445 <151> 2017-04-13 <160> 47 <170> PatentIn version 3.5 <210> 1 <211> 1152 <212> RNA <213> Measles virus <400> 1 accagacaaa gcugggaaua gaaacuucgu auuuucaaag uuuucuuuaa uauauugcaa 60 auaaugccua accaccuagg gcaggauuag gguuccggag uucaaccaau uaguccuuaa 120 ucagggcacu guauccgacu aacuuauacc auucuuuggu cuccuugacu guuaccuuaa 180 aaacccacuc acguuucaaa ccccccguca uaauaaucug uuucucugac uuggauagau 240 ucuuaacgaa gauauucugg uguaagucua guaucagaua gccggacuug agauucugga 300 uaaacuuauu uaucaacuuu cuguucccgg aguaaagaag aauuggcccc caaaauuugc 360 gggugauccu agaauauaagu ucucguugcc ugcuacuuac caauacgggg uaagcgugga 420 acaucccuug uugacuucuu ugguugucuu ugaaucuugc caacucccug uagaggauga 480 guauagaauu aagcaaucca ucuugcccug aggcaacauc augguggauc aauucuuugc 540 acagcuuagg uccguuaauu gccaacccgc aauugaucag caccugcucu auagguguaa 600 guuuuuucag aguaggauug auaucaccuc uacuaacugc gucucccaca auugcuugua 660 ugcagcuuag uugcuuaaug gauaggaugu gaccuauaag uccaggugaa guccucacag 720 augauucaau uaucugcugc uuaaucuuuu caggauucau uagccgguua gccuugagau 780 cugucauaac caaauaagau ucaguagaua ugaaguugcu guaucuaggg uauacaaggu 840 ucacuucucu auaaugagac ccuacauaac uuauaaaucc cugaacaaaa uccccgcuga 900 aaggcauaag cuuaaucacc aguauugauc cuauuuugcc caggagcaga gccaucgaua 960 agauggcugc caauuccucu agcuucucua uaguaucuuu guuaggcaag uuagucggau 1020 acagugcccu gauuaaggac uaauugguug aacuccggaa cccuaauccu gcccuaggg 1080 guuaggcauu auuugcaaua uauuaaagaa aacuuugaaa auacgaaguu ucuauuccca 1140 gcuuugucug gu 1152 <210> 2 <211> 58 <212> RNA <213> Measles virus <400> 2 ggauacagug cccugauuaa ggacuaauug guugaacucc ggaacccuaa uccugccc 58 <210> 3 <211> 79 <212> RNA <213> Measles virus <400> 3 cccggugguu aggcauuauu ugcaauauau uaaagaaaac uuugaaaaua cgaaguuucu 60 auucccagcu uugucuggu 79 <210> 4 <211> 139 <212> RNA <213> Measles virus <400> 4 cccgggauaca gugcccugau uaaggacuaa uugguugaac uccggaaccc uaauccugcc 60 cuagggugguu aggcauuauu ugcaauauau uaaagaaaac uuugaaaaua cgaaguuucu 120 auucccagcu uugucuggu 139 <210> 5 <211> 79 <212> RNA <213> Measles virus <400> 5 ggugguuagg cauuauuugc aauauauuaa agaaaacuuu gaaaauacga aguuucuauu 60 cccagcuuug ucugguccc 79 <210> 6 <211> 139 <212> RNA <213> Measles virus <400> 6 ggauacagug cccugauuaa ggacuaauug guugaacucc ggaacccuaa uccugcccua 60 ggugguuagg cauuauuugc aauauauuaa agaaaacuuu gaaaauacga aguuucuauu 120 cccagcuuug ucugguccc 139 <210> 7 <211> 99 <212> RNA <213> Measles virus <400> 7 ggaacccuaa uccugcccua ggugguuagg cauuauuugc aauauauuaa agaaaacuuu 60 gaaaauacga aguuucuauu cccagcuuug ucugguccc 99 <210> 8 <211> 110 <212> RNA <213> Measles virus <400> 8 gguugaacuc cggaacccua auccugcccu aggugguuag gcauuauuug caauauauua 60 aagaaaacuu ugaaaauacg aaguuucuau ucccagcuuu gucugguccc 110 <210> 9 <211> 119 <212> RNA <213> Measles virus <400> 9 ggacuaauug guugaacucc ggaacccuaa uccugcccua ggugguuagg cauuauuugc 60 aauauauuaa agaaaacuuu gaaaauacga aguuucuauu cccagcuuug ucugguccc 119 <210> 10 <211> 73 <212> RNA <213> Measles virus <400> 10 ggauacagug cccugauuaa ggacuaauug guugaacucc ggaacccuaa uccugcccua 60 gggguuagg cau 73 <210> 11 <211> 140 <212> RNA <213> Measles virus <400> 11 accagacaaa gcugggaaua gaaacuucgu auuuucaaag uuuucuuuaa uauauugcaa 60 auaaugccua accaccuagg gcaggauuag gguuccggag uucaaccaau uaguccuuaa 120 ucagggcacu guauccgacu 140 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN M362) <400> 12 tcgtcgtcgt tcgaacgacg ttgat 25 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2006) <400> 13 tcgtcgtttt gtcgttttgt cgtt 24 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1668) <400> 14 tccatgacgt tcctgatgct 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1826) <400> 15 tccatgacgt tcctgacgtt 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2007) <400> 16 tcgtcgttgt cgttttgtcg tt 22 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA(ODN BW006) <400> 17 tcgacgttcg tcgttcgtcg ttc 23 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2395) <400> 18 tcgtcgtttt cggcgcgcgc cg 22 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN M362 control) <400> 19 tgctgctgct tgcaagcagc ttgat 25 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN M362 control_TpC substitute) <400> 20 ttcttcttct ttcaatcatc ttgat 25 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN M362 control_CpC substitute) <400> 21 tcctcctcct tccaaccacc ttgat 25 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2006 control) <400> 22 tgctgctttt gtgcttttgt gctt 24 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2006 control_TpC substitute) <400> 23 ttcttctttt gttcttttgt tctt 24 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2006 control_CpC substitute) <400> 24 tcctcctttt gtccttttgt cctt 24 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2007 control) <400> 25 tgctgcttgt gcttttgtgc tt 22 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2007 control_TpC substitute) <400> 26 ttcttcttgt tcttttgttc tt 22 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2007 control_CpC substitute) <400> 27 tcctccttgt ccttttgtcc tt 22 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN BW006 control) <400> 28 tgcagcttgc tgcttgctgc ttc 23 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN BW006 control_TpC substitute) <400> 29 ttcatctttc ttctttcttc ttc 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN BW006 control_CpC substitute) <400>30 tccaccttcc tccttcctcc ttc 23 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2395 control) <400> 31 tgctgctttt ggggggcccc cc 22 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2395 control_TpC substitute) <400> 32 ttcttctttt gggggtcccc cc 22 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 2395 control_CpC substitute) <400> 33 tcctcctttt gggggcccccc cc 22 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1668 control) <400> 34 tccatgagct tcctgatgct 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1668 control_TpC substitute) <400> 35 tccatgatct tcctgattct 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1668 control_CpC substitute) <400> 36 tccatgacct tcctgatcct 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1826 control) <400> 37 tccatgagct tcctgagctt 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1826 control_TpC substitute) <400> 38 tccatgatct tcctgatctt 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA (ODN 1826 control_CpC substitute) <400> 39 tccatgacct tcctgacctt 20 <210> 40 <211> 165 <212> DNA <213> Artificial Sequence <220> <223> DNA/RNA chimeric molecule for sense chain <220> <221> misc_feature <222> (26)..(165) <223> RNA <400> 40 tgctgctgct tgcaagcagc ttgataccag acaaagcugg gaauagaaac uucguauuuu 60 caaaguuuuc uuuaauauau ugcaaauaau gccuaaccac cuagggcagg auuaggguuc 120 cggaguucaa ccaauuaguc cuuaaucagg gcacuguauc cgacu 165 <210> 41 <211> 140 <212> RNA <213> Artificial Sequence <220> <223> Synthetic RNA for antisense chain <400> 41 agucggauac agugcccuga uuaaggacua auugguugaa cuccggaacc cuaauccugc 60 ccuagguggu uaggcauuau uugcaauaua uuaaagaaaa cuuugaaaau acgaaguuuc 120 uauucccagc uuugucuggu 140 <210> 42 <211> 145 <212> DNA <213> Artificial Sequence <220> <223> DNA/RNA chimeric molecule for sense chain <400> 42 tgctgctgct tgcaagcagc ttgataauag aaacuucgua uuuucaaagu uuucuuuaau 60 auauugcaaa uaaugccuaa ccaccuaggg caggauuagg guuccggagu ucaaccaauu 120 aguccuuau cagggcacug uaucc 145 <210> 43 <211> 120 <212> RNA <213> Artificial Sequence <220> <223> Synthetic RNA for antisense chain <400> 43 ggauacagug cccugauuaa ggacuaauug guugaacucc ggaacccuaa uccugcccua 60 ggugguuagg cauuauuugc aauauauuaa agaaaacuuu gaaaauacga aguuucuauu 120 <210> 44 <211> 145 <212> DNA <213> Artificial Sequence <220> <223> DNA/RNA chimeric molecule for sense chain <400> 44 tgctgctgct tgcaagcagc ttgataauag aaacuucgua uuuucaaagu uuucuuuaau 60 auauuaauag aaacuucgua uuuucaaagu uuucuuuaau auauuaauag aaacuucgua 120 uuuucaaagu uuucuuuaau auauu 145 <210> 45 <211> 120 <212> RNA <213> Artificial Sequence <220> <223> Synthetic RNA for antisense chain <400> 45 aauauauuaa agaaaacuuu gaaaauacga aguuucuauu aauauauuaa agaaaacuuu 60 gaaaauacga aguuucuauu aauauauuaa agaaaacuuu gaaaauacga aguuucuauu 120 <210> 46 <211> 155 <212> DNA <213> Artificial Sequence <220> <223> DNA/RNA chimeric molecule for sense chain <400> 46 tgctgctgct tgcaagcagc ttgataccag acaaagcugg gaauagaaac uucguauuuu 60 caaaguuuuc uuuaauauau ugcaaauaau gccuaaccac cuagggcagg auuaggguuc 120 cggaguucaa ccaauuaguc cuuaaucagg gcacu 155 <210> 47 <211> 130 <212> RNA <213> Artificial Sequence <220> <223> Synthetic RNA for antisense chain <400> 47 agugcccuga uuaaggacua auugguugaa cuccggaacc cuaauccugc ccuaggguggu 60 uaggcauuau uugcaauaua uuaaagaaaa cuuugaaaau acgaaguuuc uauucccagc 120 uuugucuggu 130

Claims (1)

Treatment method.

Images